ERN1 Knockdown Modifies the Impact of Glucose and Glutamine Deprivations on the Expression of EDN1 and Its Receptors in Glioma Cells

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72 ENDOCRINE REGULATIONS, Vol. 55, No. 2, 72–82, 2021

doi:10.2478/enr-2021-0009

ERN1 knockdown modifies the impact of glucose and glutamine deprivations on the expression of EDN1 and its receptors in glioma cells

Dmytro O. Minchenko1,2, Olena O. Khita1, Dariia O. Tsymbal1, Yuliia M. Viletska1, Myroslava Y. Sliusar1, Yuliia V. Yefimova1, Liudmyla O. Levadna2, Dariia A. Krasnytska1, Oleksandr H. Minchenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine; 2National Bogomolets Medical University, Kyiv, Ukraine E-mail: [email protected]

Objective. The aim of the present investigation was to study the impact of glucose and glutamine deprivations on the expression of genes encoding EDN1 (endothelin-1), its cognate receptors (EDNRA and EDNRB), and ECE1 (endothelin converting enzyme 1) in U87 glioma cells in response to knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), a major signaling pathway of endoplasmic reticulum stress, for evaluation of their possible implication in the control of glioma growth through ERN1 and nutrient limitations. Methods. The expression level of EDN1, its receptors and converting enzyme 1 in control U87 glioma cells and cells with knockdown of ERN1 treated by glucose or glutamine deprivation by quantitative polymerase chain reaction was studied. Results. We showed that the expression level of EDN1 and ECE1 genes was significantly up- regulated in control U87 glioma cells exposure under glucose deprivation condition in comparison with the glioma cells, growing in regular glucose containing medium. We also observed up- regulation of ECE1 gene expression in U87 glioma cells exposure under glutamine deprivation as well as down-regulation of the expression of EDN1 and EDNRA mRNA, being more significant for EDN1. Furthermore, the knockdown of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose and glutamine deprivation conditions. Thus, the ERN1 knockdown led to a strong suppression of EDN1 gene expression under glucose deprivation, but did not change the effect of glutamine deprivation on its expression. At the same time, the knockdown of ERN1 signaling introduced the sensitivity of EDNRB gene to both glucose and glutamine deprivations as well as completely removed the impact of glucose deprivation on the expression of ECE1 gene. Conclusions. The results of this study demonstrated that the expression of endothelin-1, its receptors, and ECE1 genes is preferentially sensitive to glucose and glutamine deprivations in gene specific manner and that knockdown of ERN1 significantly modified the expression ofEDN1, EDNRB, and ECE1 genes in U87 glioma cells. It is possible that the observed changes in the expression of studied genes under nutrient deprivation may contribute to the suppressive effect of ERN1 knockdown on glioma cell proliferation and invasiveness.

Key words: ERN1 knockdown, mRNA expression, EDN1, EDNRA, EDNRB, ECE1, glucose and glutamine deprivations, U87 glioma cells

Corresponding author: Oleksandr H. Minchenko, Prof, Department of Molecular Biology, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Leontovycha 9, Kyiv 01030, Ukraine; e-mail: [email protected]. Minchenko, et al. 73

The unfolded protein response is a highly tial for this purpose (Huber et al. 2013; Parzych et al. conserved protein quality control mechanism, acti- 2019). Glucose as well as glutamine are important vated in response to endoplasmic reticulum stress. substrates for glycolysis and glutaminolysis, which The growing tumor requires the endoplasmic retic- are important to glioma development and a more ulum stress for own neovascularization and growth aggressive behavior through regulation of the cell as well as for inhibition of apoptosis under stressful cycle at distinct stages (Colombo et al. 2011). environmental conditions (Drogat et al. 2007; Auf It is interesting to note that endoplasmic reticulum et al. 2010; Doultsinos et al. 2017). The endoplasmic stress and glucose as well as glutamine level are very reticulum stress signaling represents an integrated important and complementary factors for tumor signal transduction pathway that transmits infor- growth and that ERN1 mediated stress signaling can mation about the protein folding status at the endo- significantly modify the effects of glutamine and plasmic reticulum to the nucleus and cytosol to glucose deprivations as well as hypoxia on numerous restore proteostasis (Bravo et al. 2013; Chevet et al. gene expressions (Minchenko et al. 2014, 2016, 2017; 2015; Lhomond et al. 2018; Marciniak 2019). Malig- 2019; Zhao et al. 2017; Tsymbal et al. 2020). However, nant tumors use the unfolded protein response the detailed molecular mechanisms of the interaction and its signaling pathways to augment cancer cells of hypoxia and glucose deprivation with ERN1 medi- proliferation (Dejeans et al. 2015; Manie et al. 2014; ated stress signaling pathway are complex yet and Papaioannou et al. 2018). It is interesting to note that warrant further study. stimulation of ERN1/IRE1 (endoplasmic reticulum The peptide hormone endothelin-1 (EDN1; to nucleus signaling 1/inositol requiring enzyme 1) ET-1) and its related receptors, such as EDNRA and division of the endoplasmic reticulum stress response EDNRB, play an important role not only in the main- is tightly linked to apoptosis and cell death, and inhi- tenance of vascular tone, but they are also localized bition of its functional activity has been demon- to non-vascular structures including epithelial cells, strated to result in substantial anti-proliferative effect glia and neurons and play multiple, complex roles in glioma growth (Auf et al. 2010, 2013; Johnson et al. (Dashwood and Loesch 2009; Khimji and Rockey 2011; Minchenko et al. 2014, 2015c; Markouli et al. 2010; Stow et al. 2011). Endothelin-1 and its recep- 2020). Additionally, suppression of ERN1 endoribo- tors also have co-mitogenic activity, potentiating the nuclease has more robust anti-proliferative influence effects of other growth factors such as PDGF and have on glioma cells and preferentially different impact on been linked to variable diseases including diabetes, the expression of some ERN1-related pathway genes traumatic shock, and tumorigenesis (Minchenko et (Auf et al. 2013; Minchenko et al. 2015c). al. 1999, 2003; Dashwood and Loesch 2009; Khimji Glioblastoma multiforme is the most common and Rockey 2010; Stow et al. 2011; Palmer et al. 2012; and malignant type of primary brain tumor of the Cook et al. 2015; Dojo Soeandy et al. 2019). There are central nervous system with poor prognosis and high also data indicating that EDN1 is a neuropeptide, mortality. The current clinical treatments for glioblas- which is implicated in a number of neural circuits toma consist of tumor resection, radiotherapy, and where its transmitter affects range from a role in chemotherapy (Nayak and Reardon 2017; Almanza et pain and temperature control to its action on the al. 2019; Yang et al. 2020). The rapid growth of cancers hypothalamo-neurosecretory system (Dashwood generates microenvironmental changes in regards to and Loesch 2009). Recently, it was shown that mRNA nutrient deprivation, thus inducing the activation of levels encoding EDN1 and its receptors, known to be endoplasmic reticulum stress signaling pathways, elevated in amyotrophic lateral sclerosis, were sharply cell proliferation, and survival (Obacz et al. 2017; increased by knockdown of C9 (C9ORF72) gene, Zhao et al. 2017; Obiedat et al. 2019; Liu et al. 2020). which encodes a protein that functions in control Furthermore, a better knowledge of tumor responses of endothelin and glutamate signaling (Fomin et al. to nutrient deprivation is required to elaborate better 2018). Differential and tissue-specific production of therapeutical strategies of cell sensibilization, based EDN1 must be tightly regulated in order to preserve on the blockade of survival mechanisms (Iurlaro et these biologically diverse actions. al. 2017; Riabovol et al. 2019; Teramoto and Katoh It is interesting to note that the palmitic acid 2019; Minchenko et al. 2020a, b; Ratushna 2020). increases ET-1 expression in endothelial cells through Cell proliferation and apoptosis are strongly depen- the induction of endoplasmic reticulum stress and dent on glycolysis and glucose level, because there is the activation of protein kinase C, providing a novel the molecular connection between metabolism, cell mechanistic insight into the pathogenesis of obesity- cycle progression, and the delivery of nutrients essen- associated hypertension and cardiovascular diseases 74 ERN1 knockdown modifies the impact of nutrient deficits on EDN1 family genes including atherosclerosis (Zhang et al 2018). The hypoxia responsible. Its promoter and intron regions primary mechanism thought the control of EDN1 contain the HIF binding sites (Khamaisi et al. 2015). bioavailability is the rate of transcription from It has also been shown that endothelin-1 produc- the EDN1 gene. Studies conducted on a variety of tion is controlled by TIMAP-protein phosphatase cell types have identified key transcription factors 1-complex via ECE1 dephosphorylation (Boratko et including HIF that govern EDN1 expression (Aversa al. 2016). et al. 1997; Minchenko and Caro 2000; Stow et al. The aim of this study was to examine the impact of 2011). glucose and glutamine deprivations on the expression The endothelin receptor type A plays a role in of genes encoded EDN1, its receptors, and converting potent and long-lasting vasoconstriction and cell enzyme 1 in U87 glioma cells in response to knock- proliferation. Thus, Arabanian et al. (2018) have down of ERN1 activity for evaluation of their possible shown that overexpression of EDNRA led to an significance in the control of glioma growth through increased proliferation and resistance to apoptosis endoplasmic reticulum stress signaling mediated by of bone marrow cells in vitro and that MEIS1 binds IRE1 and nutrient limitation. to the EDNRA promoter region, suggesting a regu- latory role for MEIS1 in endothelin-1 receptor type Materials and methods A expression and in HOXA9/MEIS1-driven leuke- mogenesis. Furthermore, the endothelin axis (EDN1 Cell lines and culture conditions. The glioma cell and EDN1 receptor A) is involved in cellular growth, line U87 was obtained from ATCC (USA) and grown differentiation, cell motility, invasiveness, and tumor in high glucose (4.5 g/l) Dulbecco’s modified Eagle’s progression as well as drug resistance in several minimum essential medium (Gibco, Invitrogen, cancers. Polymorphisms of these genes increase the Carlsbad, CA, USA) supplemented with glutamine risk of papillary thyroid cancer developing (Aydin et (2 mM), 10% fetal bovine serum (Equitech-Bio, Inc., al. 2019; Pulido et al. 2020). Recently, it was observed USA), penicillin (100 units/ml; Gibco) and strepto- an abnormal expression of endothelin receptor mycin (0.1 mg/ml; Gibco) at 37oC in incubator with type B (EDNRB) in hepatocellular carcinoma and 5% CO2. In this work, we used two sublines of these confirmed its potential clinical significance (Zhang cells, which were described previously (Auf et al. et al. 2019). Thus, endothelin-1 receptor blockade 2010, 2013). One subline was obtained by selection is a new possible therapeutic approach in ovarian of stable transfected clones with overexpression of cancer and multiple myeloma, because the majority empty vector pcDNA3.1, which was used for creation of multiple myeloma cell lines and primary malig- of dnERN1. This untreated subline of glioma cells nant plasma cells express high levels of EDNRA (control glioma cells) was used as control 1 in the and EDNRB and release EDN1 (Rosano et al. 2017; study of the effect of glucose and glutamine depri- Russignan et al. 2017). vations on the expression level of genes encoding The endothelin-converting enzyme-1 (ECE1) EDN1, its receptors (EDNRA, EDNRB), and endo- is also involved in proteolytic processing of endothelin converting enzyme ECE1. Second subline was thelin precursor to biologically active peptide. Active obtained by selection of stable transfected clones EDN1 has a short half-life, so its biological effects are with overexpression of ERN1 dominant/negative completely dependent on its enzymatic activation by construct (dnERN1), having suppression of both the ECE1. There are data indicating that expression of protein kinase and endoribonuclease activities of this ECE1 gene is elevated in several tumors and cancer signaling protein (Auf et al. 2010). All used sublines cell lines and that this protein has relation to cancer of glioma cells in this study are grown in the presence cell proliferation and invasiveness (Niechi et al. 2015; of geneticin (G418), while these cells carrying empty Tapia and Niechi 2019). Furthermore, Miners and vector pcDNA3.1 or dnERN1 construct. Glucose and Love (2017) have shown that endothelin-converting glutamine deprivation conditions were created by enzymes degrade alpha-synuclein and are reduced in changing the complete DMEM medium into culture dementia with Lewy bodies. Thus, it participates in plates on DMEM medium without glucose or gluta- the metabolism of α-synuclein that could contribute mine and plates were exposed to this condition for to the development and progression of dementia. It 16 h. is interesting to note that miR-199a-5p, which nega- RNA isolation. Total RNA was extracted from tively regulated ECE1, and silencing the ECE1 gene glioma cells using the Trizol reagent according to are protected the rat spinal cord injury after isch- manufacturer’s protocol (Invitrogen, Carlsbad, emia-reperfusion (Bao et al. 2018). The ECE1 gene is CA, USA). The RNA pellets were washed with 75% Minchenko, et al. 75 ethanol and dissolved in nuclease-free water. For deprivation, is significantly up-regulated (+57%) in additional purification RNA samples were re-precip- comparison with non-treated cells. At the same time, itated with 95% ethanol and re-dissolved again in inhibition of ERN1 signaling completely modifies nuclease-free water. RNA concentration and spectral the effect of glucose deprivation on the expression characteristics were measured using NanoDrop One of endothelin-1 gene expression (Figure 1). Thus, Spectrophotometer (Thermo Scientific). the exposure of these cells under glucose depriva- Reverse transcription and quantitative PCR tion leads to a strong down-regulation (–68%) of analysis. The impact of glucose and glutamine depri- EDN1 mRNA expression. We next investigated the vations on the expression level of endothelin-1 and its effect of ERN1 knockdown on the expression of gene receptors (EDNRA and EDNRB) and ECE1 mRNAs encoding endothelin receptor type A in glioma cells as well as ACTB as housekeeping gene were measured treated by glucose deprivation. As shown in Figure 2, in control U87 glioma cells and cells with a deficiency the expression level of EDNRA mRNA is not signifi- of ERN1, introduced by dnERN1, by quantitative cantly changed in both control and ERN1 knock- polymerase chain reaction using SYBRGreen Mix down U87 glioma cells in comparison with cells, (ABgene, Thermo Fisher Scientific, Epsom, Surrey, growing with glucose. Furthermore, similar results UK) and “QuantStudio 5 Real-Time PCR System” in the expression of EDNRB gene we received in (Applied Biosystems, USA). Thermo Scientific Verso control glioma cells (Figure 3). However, inhibition cDNA Synthesis Kit (Germany) was used for reverse of ERN1 functional activity by dnERN1 introduces transcription as described previously (Minchenko et sensitivity of EDNRB gene expression to glucose- al. 2019). Polymerase chain reaction was performed limited condition and leads to small but statistically in triplicate. The expression of beta-actin mRNA significant up-regulation (+12%) of this gene expres- was used as control of analyzed RNA quantity. The sion as compared to appropriate control (dnERN1) pair of primers specific for each studied gene was glioma cells (Figure 3). received from Sigma-Aldrich (St. Louis, MO, U.S.A.) We also studied the expression of gene encoding and used for quantitative polymerase chain reaction endothelin converting enzyme 1, which play an as described previously by Minchenko et al. (2019). Quantitative PCR analysis was performed using a special computer program “Differential expression calculator” and statistical analysis using Excel program and OriginPro 7.5 software as described previously (Bochkov et al. 2006; Rudnytska et al. 2021). Comparison of two means was performed by the use of two-tailed Student’s t-test. The value p<0.05 was considered significant in all cases. The values of EDN1, EDNRA, EDNRB, and ECE1 gene expressions were normalized to the expression of beta-actin mRNA and represented as percent of control (100%). All values are expressed as mean ± SEM from triplicate measurements performed in 4 independent experiments. The amplified DNA fragments were also analyzed on a 2% agarose gel and that visualized by SYBR* Safe DNA Gel Stain (Life Technologies, Carlsbad, CA, USA).

Results

The possible role of endoplasmic reticulum stress signaling mediated by ERN1 signaling protein in the control of endothelin-1, its receptors, and endo- Figure 1. Impact of glucose deprivation on the expression level thelin converting enzyme 1 gene expressions in U87 of endothelin 1 (EDN1) gene expression in control U87 glioma cells (Vector) and cells with a blockade of the ERN1 (dnERN1) glioma cells was studied. As shown in Figure 1, the measured by qPCR. Values of EDN1 mRNA expression were expression of EDN1 mRNA in control U87 glioma normalized to the level of beta-actin mRNA and represented as cells (transfected by empty vector), treated by glucose percent of control 1 (100 %); n=4. 76 ERN1 knockdown modifies the impact of nutrient deficits on EDN1 family genes

Figure 2. Impact of glucose deprivation on the expression level Figure 3. Impact of glucose deprivation on the expression level of endothelin receptor type A (EDNRA) gene expression level of endothelin receptor type B (EDNRB) gene expression level in control U87 glioma cells (Vector) and cells with a blockade in control U87 glioma cells (Vector) and cells with a blockade of the ERN1 (dnERN1) measured by qPCR. Values of EDNRA of the ERN1 (dnERN1) measured by qPCR. Values of EDNRB mRNA expression were normalized to the level of beta-actin mRNA expression were normalized to beta-actin mRNA level mRNA and represented as percent of control 1 (100%); n=4. and represent as percent for control 1 (100%); n=4.

important role in the regulation of endothelin-1 control cells (dnERN1) (–64%; Figure 5). At the same activity and showed its strong sensitivity to glucose time, the impact of glutamine deprivation on the deprivation (Figure 4). Thus, the expression level expression of gene encoding endothelin receptor type of ECE1 gene is significantly up-regulated (+133%) A and B genes in control glioma cells was different in glioma cells without both ERN1 protein kinase (Figures 6 and 7). Thus, the exposure of control and endoribonuclease activities in comparison with glioma cells under glutamine-limited condition cells, transfected by empty vector. At the same time, leads to down-regulation of EDNRA gene expression knockdown of ERN1 signaling protein completely (–20%) but EDNRB gene expression was resistant to eliminated the sensitivity of ECE1 gene expression to this experimental condition. It is interesting to note glucose-limited condition (Figure 4). that inhibition of ERN1 signaling enzyme function We next investigated the impact of glutamine modifies effect of glutamine deprivation on endo- deprivation on the expression of genes encoding thelin receptor type B gene expression and leads to endothelin-1 and related proteins in U87 glioma cells up-regulation of the expression of this endothelin in relation to inhibition of ERN1 signaling protein receptor gene (+38%) in comparison to appropriate function. It was shown that in control glioma cells control cells (Figure 7). As shown in Figure 8, the (transfected by empty vector) the expression level of exposure of control glioma cells under glutamine- EDN1 gene is strongly down-regulated (–63%) under limited condition leads to significant up-regulation glutamine-limited condition in comparison with of endothelin converting enzyme 1 gene expression cells growing in regular DMEM medium (Figure 5). (+37%) in comparison with control cells growing with Furthermore, inhibition of ERN1 signaling enzyme glutamine-containing medium. At the same time, function does not significantly change the sensi- the inhibition of ERN1 functional activity decreases tivity of endothelin-1 gene expression to glutamine the sensitivity of ECE1 gene expression to glutamine deprivation condition as compared to corresponding deprivation. Therefore, the expression level of ECE1 Minchenko, et al. 77

Figure 4. Impact of glucose deprivation on the expression level Figure 5. Impact of glutamine deprivation on the expression of endothelin converting enzyme 1 (ECE1) gene expression level of EDN1 mRNA in control U87 glioma cells (Vector) and level in control U87 glioma cells (Vector) and cells with a block- ERN1 knockdown glioma cells (dnERN1) measured by qPCR. ade of the ERN1 (dnERN1) measured by qPCR. Values of ECE1 Values of EDN1 mRNA expression were normalized to beta- mRNA expression were normalized to beta-actin mRNA level actin mRNA level and represented as percent for control 1 and represented as percent for control 1 (100%); n=4. (100%); n=4. gene in ERN1 knockdown glioma cells growing under glutamine deprivation condition is also increased but to a much lesser extent (+18%; Figure 8). Thus, the glucose and glutamine deprivation affects the expression of most studied genes but in different ways and preferentially in ERN1 dependent manner.

Discussion

In this work, we studied the impact of glucose and glutamine deprivation on the expression of genes encoding endothelin-1, its receptors (EDNRA and EDNRB) and endothelin converting enzyme 1, which have relation to functional activity of EDN1, in relation to inhibition of ERN1, the major signaling pathway of the unfolded protein response, in U87 glioma cells with ERN1 knockdown, introduced by dnERN1. Results of these experiments are summa- rized in Figure 9. These data are important for evaluation of possible significance of these genes in the control of glioma growth through endoplasmic Figure 6. Impact of glutamine deprivation on the expression reticulum stress signaling mediated by ERN1, level of EDNRA mRNA in control U87 glioma cells (Vector) and ERN1 knockdown glioma cells (dnERN1) measured by because this stress signaling pathway is involved qPCR. Values of this mRNA expression were normalized to in numerous metabolic pathways and inhibition of beta-actin mRNA level and represented as percent for control ERN1 signaling enzyme activity in glioma cells had 1 (100%); n=4. 78 ERN1 knockdown modifies the impact of nutrient deficits on EDN1 family genes

Figure 7. Impact of glutamine deprivation on the expression Figure 8. Impact of glutamine deprivation on the expression level of EDNRB mRNA in control U87 glioma cells (Vector) level of ECE1 mRNA in control U87 glioma cells (Vector) and and ERN1 knockdown glioma cells (dnERN1) measured by ERN1 knockdown glioma cells (dnERN1) measured by qPCR. qPCR. Values of EDNRB mRNA expression were normalized Values of this mRNA expression were normalized to beta-actin to beta-actin mRNA level and represented as percent for con- mRNA level and represented as percent for control 1 (100%); trol 1 (100%); n=4. n=4.

clear anti-tumor effects (Auf et al. 2010, 2013; Bravo et al. 2013; Manie et al. 2014; Minchenko et al. 2013, 2015a, b; Logue et al. 2018). Recently, we showed that the expression of EDN1, its receptors, and ECE1 genes is responsible to endoplasmic reticulum stress signaling mediated by ERN1. However, molecular mechanisms of changes in these genes’ expression are different (Minchenko et al. 2019). Thus, the expression of EDNRA and ECE1 genes is regulated by ERN1 endoribonuclease, but EDN1 and EDNRB genes – preferentially by ERN1 protein kinase through mechanisms described by Auf et al. (2013) and Minchenko et al. (2015c). Endoplasmic reticulum stress dependent regulation of EDN1 and related proteins agree well with its involving in cancer biology as well as in many other important processes (Teoh et al. 2014; Cook et al. 2015; Niechi et al. 2015; Tapia and Niechi 2019; Minchenko and Caro 2000; Minchenko et al. 2003). At the same time, the prominent pathophysiological aspects of the endothelin system in glioma cells as well as in astrocytes are not clear yet. Figure 9. Schematic demonstration of glucose (A) and glutamine deprivations (B) on EDN1, EDNRA, EDNRB, and ECE1 We also showed that glucose deprivation enhanced genes expression profile in control and ERN1 knockdown glio- the expression of EDN1 and ECE1 genes and that ma cells; NS – no significant changes. knockdown of ERN1 signaling enzyme function Minchenko, et al. 79 significantly modified the response of these genes to sion of EDN1 gene and significantly lesser – on glucose-limited condition (Figure 9A). It is possible ENDRA gene expressions both in control and ERN1 that the ERN1 signaling is very important for induc- knockdown glioma cells (Figure 9B). It is possible tion of these genes’ expression under glucose depri- that this effect of glutamine-limited condition on vation and in this connection the knockdown of EDN1 and EDNRA gene expressions reflects the ERN1 removes sensitivity of ECE1 gene to glucose- sensitivity of these genes to glutamine supply. Thus, limited condition and leads to a strong suppression these results validate tight interaction of endo- of EDN1 gene expression at this experimental state. plasmic reticulum stress signaling with the delivery At the same time, the expressions of both endothelin of essential nutrients such as glutamine and glucose, receptors are resistant to glucose-limited condition. which are important substrates for glycolysis and These data clearly demonstrate that glucose-depen- glutaminolysis, as well as glioma growth (Colombo dent regulation of endothelin system is preferentially et al. 2011; Huber et al. 2013). However, precise realized through EDN1 and ECE1 gene expressions. molecular mechanisms are not clear yet and warrant It is possible that glucose limitation affects different further investigations. signaling pathways of endoplasmic reticulum stress This study provides unique insights into the and thus changes the ERN1 signaling, because there molecular mechanisms regulating the expression of are data that glucose deprivation alters the proapop- genes encoding endothelin-1, its receptors, and endo- totic PERK-CHOP/GADD153 pathway as well as thelin converting enzyme 1 in glioma cells under ATF4-mediated apoptosis (Huber et al. 2013; Iurlaro glucose and glutamine deprivations in response to et al. 2017). Furthermore, there are data that glucose inhibition of ERN1 activity, attesting to the fact that deprivation condition can augment the sensitivity of endoplasmic reticulum stress is a necessary compo- cancer cells to anti-cancer drugs, particularly arcti- nent of malignant tumor growth. In this regards, genin, which inhibits the growth of numerous cancer endoplasmic reticulum stress signaling pathways are cells and induces tumor cell death under glucose- important targets in combined strategies for glio- limited condition, possibly by blocking the unfolded blastoma therapy (Johnson et al. 2011; Festuccia et al. protein response (He et al. 2018). Results of our study 2020; Liu et al. 2020; Markouli et al. 2020). clarify possible mechanisms of glucose deprivation on the proliferation of ERN1 knockdown glioma cells Acknowledgement through specific changes in the expression of genes encoding endothelin-1 and related proteins. This work was funded by the State Budget Program At the same time, we observed a strong suppres- “Support for the Development of Priority Areas of sive impact of glutamine deprivation on the expres- Scientific Research” (Code: 6541030).

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